An epidermal growth factor (EGF) has been first found by S. Cohen in U.S.A. in 1953. An EGF derived from human is polypeptide composed of 53 amino acids, which has a molecular weight of 6,045 Dalton and three disulfide bonds. It is known that since the EGF has activities such as suppression of gastric acid secretion, acceleration of cell growth, and acceleration of mitosis to various cells including a mesenchymal cell and an epithelial cell of mammals, the EGF can be used as an agent for treating a stomach ulcer and an agent for treating wound of skin or cornea. Recently, the EGF is being used as an agent for treating wound and the damaged stomach walls, and also is being recognized as a medicine for diabetic foot ulcer that mainly occurs in a diabetic.
However, in recent years, the field that uses an EGF the most is the field of cosmetics. With advancing years, the concentrations of growth factors, such as an EGF, are lower and the functions of cell recycle and cell division are deteriorated, thereby accelerating the ageing process, for example, forming wrinkles and reducing elasticity. In general, for the skin after 25 years of age, the growth factors are decreased, and thus metabolism or cell recycle ability is gradually getting late, thereby forming wrinkles. The cycle of skin regeneration for healthy young people is about 4 weeks, but the cycle of skin regeneration for the skin after 25 years of age is 6 weeks, that is late. Therefore, the generation ability of skin cells is decreased and stratum corneum is thickened, thereby proceeding the ageing process. An EGF is a factor for accelerating the growth of an epithelial cell, and it is known that the growth of an epithelial cell is accelerated by inducing the growth and division of cells, especially, an epithelial cell and skin cell of mammal by transferring signal through an EGF receptor present on a cell membrane. Therefore, when an EGF is applied to the skin using cosmetics, the EGF plays a key role for the skin regeneration, for example, the proliferations of an epithelial cell and hypodermal cell are accelerated, the cell proliferation of fibroblasts synthesizing collagen that is a component constituting the dermis, vascularization of the damaged skin part is accelerated, the secretions of other regeneration stimulating factors are induced, the skin tissue takes its course in arrangement, the synthesis of fibronectin that is a material allowing a net to be formed is accelerated, and the scar in the wounded area is minimized. Therefore, the EGF can help the original functions of the skin that are deteriorated with advancing years and can accelerate the growth of the new skin cells. For this reason, the EGF was designated on International Cosmetic Ingredient Dictionary (ICID) of The Cosmetic, Toiletry and Fragrance Association (CTFA), and recently, also was approved as a cosmetic raw material at Korea Food & Drug Administration. Therefore, the EGF can be formally used as a cosmetic raw material at home and abroad.
For this reason, an EGF is tried to use in many fields. However, in early days, the EGF is obtained through purification among various growth factors derived during a generation process of stem cells, and thus its purity and activity are low, the productivity thereof is low, and it is produced through a high cost process. Therefore, the application thereof is limited due to a high cost. However, recently, it is possible to produce an EGF in bulk with low cost by producing the same protein as an EGF in the human body from microorganisms by a genetic engineering technique, and then purely isolating only an EGF using a protein isolation and purification technique.
However, the EGF thus produced is sensitive when it is present at a relatively high temperature and in an aqueous solution, and thus the activity thereof is greatly deteriorated in a general product state or under a distribution environment. Therefore, in order to use the EGF as a cosmetic raw material, the need to stabilize the EGF has been proposed. Accordingly, it is needed that an EGF mutant capable of replacing for the conventional EGF is prepared thereby providing an EGF having excellent stability while the activity thereof is maintained. Currently, a study on an EGF mutant is focused on the development of the mutant exhibiting the higher activity rather than the stability. As a case of increasing stability, there is a method of increasing a degree of EGF stability through secretion suppression by substituting unstable aspartic acid with other amino acids, but a follow-up study is not progressed (Nascimento, G. C. et al., Biochemistry. 29:9584-9591, 1990). Otherwise, there is a case of increasing stability by attaching other materials to an EGF, such as albumin fusion epidermal growth factor, PEG-EGF (Park, J. W. et al., Biochem Biophys Res Commun. 366:769-774, 2008). However, for such a case, there is a problem in that the size of the whole proteins becomes larger, and thereby it is difficult to be absorbed to the skin. Therefore, it is important to develop an EGF mutant having increased stability by only the change of amino acid in the EGF without changing the size of protein.
Meanwhile, Korean Patent No. 10-0110123 relates to a stable epidermal growth factor (EGF) composition, in which the biological activity thereof can be maintained and also the EGF composition is stable biologically, chemically, and physically by adding the additives selected from phenol, polyethylene glycol, fatty acid salts, and the like to an epidermal growth factor (EGF). However, it does not disclose the EGF mutant having high stability, which is produced by changing the amino acid sequence in the EGF according to the present invention.